Glycoconjugate vaccines

ABSTRACT

The disclosure relates to a glycoconjugate vaccine conferring protection against  Francisella tularensis  infections and a method to manufacture a glycoconjugate antigen.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Application No. 14/655,210filed Jun. 24, 2015, now U.S. Pat. No. 9,642,902, which is the U.S.National Stage of International Application No. PCT/GB2014/050159, filedJan. 21, 2014, which was published in English under PCT Article 21(2),which in turn claims the benefit of United Kingdom Application No.1301085.5, filed Jan. 22, 2013.

FIELD OF THE INVENTION

The disclosure relates to a glycoconjugate vaccine conferring protectionagainst Francisella tularensis infections and a method to manufacture aglycoconjugate antigen.

BACKGROUND TO THE INVENTION

Vaccines protect against a wide variety of infectious diseases. Manyvaccines are produced by inactivated or attenuated pathogens which areinjected into a subject, whereas others, so called ‘subunit vaccines’,are made from proteins or polysaccharides displayed on the surface ofthe pathogen. Subunit vaccines are preferred over inactivated orattenuated pathogens as they tend to cause fewer side effects. However,the development and production of a subunit vaccine requires theidentification and isolation of protective antigens from the pathogenicorganism, and moreover subunit vaccines based on polysaccharide antigensinvoke often a T-cell independent immune response which results in lowantibody titre, short half-life of the antibodies and low affinity for aspecific antigen.

The development of subunit vaccines is an active research area and ithas been recognized that the immunogenicity of polysaccharide antigenscan be enhanced by conjugation to a protein carrier. Glycoconjugateshave the ability to induce both humoral and adaptive immune responses.Currently licensed human glycoconjugate vaccines include those againstHaemophilus influenzae, Neisserria meningitidis and Streptococcuspneumoniae, by which bacterial polysaccharides are chemically bound tocarrier proteins. The H. influenzae type B (Hib) vaccine or Prevnar®, a13-valent capsule-based glycoconjugate vaccine protective againstdiseases caused by S. pneumonia, employs the carrier protein iCRM197, anon-toxic version of diphtheria toxin isolated from Corynebacteriumdiphtheria.

Although these vaccines are effective, their production requires boththe purification of polysaccharide glycan from the native pathogen andthe chemical coupling of the sugar to a suitable protein carrier, whichcan lead to impure products, low yields, variation between batches ofconjugates and poses a biohazard as the handling of the pathogenicorganism is unavoidable. This process is highly costly, inefficient andtime consuming. The use of organic systems represents a more rapid andeconomical method for the production of glycoconjugates.

So far several pathogenic bacteria have been identified formingglycoproteins and the genes involved identified. The gram negativepathogenic bacterium Campylobacter jejuni harbours a gene clusterinvolved in the synthesis of lipo-oligosaccharides and N-linkedglycoproteins. The protein glycosylation locus, a cluster of 12 genescomprising pgIA-pgIG, is involved in the glycosylation of over 30glycoproteins. Part of the gene cluster is PgIB, anoligosaccharyltransferase catalysing the transfer of glycans on to awide range of different non-species related protein acceptors,indicating broad substrate specificity. Moreover PgIB when expressed inE. coli has been used to produce novel glycoconjugates providing agenetic tool to express heterologous recombinant glycoproteins.Production of glycoconjugate vaccines in a bacterial system aredisclosed in patent application WO2009/104074. Glycoconjugatescomprising a protein carrier and an antigenic polysaccharide O-antigenform Shigella, E. coli and Pseudomonas aeruginosa using theoligosaccharyltransferase PgIB were produced, and bioconjugates againstthe Shigella O1 polysaccharide were shown to elicit an immune response.

Tularemia, also known as lemming or rabbit fever, is common in wildrodents and can be passed on to humans by contact with infected animaltissues, ticks or biting flies, or by inhalation of the infectiousorganism. Tularemia is found in North America, parts of Europe and Asiaand is caused by the gram-negative coccobacillus Francisella tularensis.F. tularensis is highly infectious, with a mortality rate of up to 30%and based on the above listed characteristics classified as a Class Abioterrorism agent. Tularemia is difficult to diagnose, however, can betreated although with varying success with antibiotics. There are novaccines available yet.

Recent studies suggest a protective effect using purifiedlipopolysaccharide (LPS) comprising an O-antigen from F. tularensis in amurine infection model; however, the development of glycoproteinvaccines protecting against highly infectious pathogens to highquantities using current methods are associated with high safetyconcerns.

We disclose a novel bacterial protein glycan coupling technology (PGCT)that allows the production of protective vaccines from the highlyvirulent wild-type strain of F. tularensis holarctica. The recombinantglycoconjugate was easily purified and was capable of providingsignificant protection against subsequent challenge when compared to LPSbased vaccine treatments.

STATEMENT OF INVENTION

According to an aspect of the invention there is provided a vaccinecomposition comprising: a carrier polypeptide comprising one or moreT-cell dependent epitopes and one or more amino acid sequences havingthe amino acid motif D/E-X-N-X-S/T wherein X is any amino acid exceptproline and crosslinked to said carrier polypeptide an antigenicpolysaccharide wherein the polysaccharide is isolated from Francisellaand is an O-antigen.

According to an aspect of the invention there is provided an immunogeniccomposition comprising: a carrier polypeptide comprising one or moreT-cell dependent epitopes and one or more amino acid sequences havingthe amino acid motif D/E-X-N-X-S/T wherein X is any amino acid exceptproline and crosslinked to said carrier polypeptide is an antigenicpolysaccharide wherein the polysaccharide is isolated from Francisellaand is an O-antigen.

In a preferred embodiment of invention said O-antigen comprises4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-QuiNAc-(1-2)-β-D-Qui4NFm-(1-),where GalNAcAN is 2-acetamido-2-deoxy-O-D-galact-uronamide, 4NFm is4,6-dideoxy-4-formamido-D-glucose and the reducing end group QuiNAc is2-acetamido-2,6-dideoxy-O-D-glucose.

In a preferred embodiment of the invention said protein carriercomprises an amino acid sequence as set forth in SEQ ID NO: 1.

In a preferred embodiment of the invention said protein carriercomprises an amino acid sequence as set forth in SEQ ID NO: 2.

In a preferred embodiment of the invention said protein carriercomprises an amino acid sequence as set forth in SEQ ID NO: 3.

In a preferred embodiment of the invention said protein carriercomprises an amino acid sequence as set forth in SEQ ID NO: 4.

In a preferred embodiment of the invention said protein carriercomprises an amino acid sequence as set forth in SEQ ID NO: 5.

In a preferred embodiment of the invention said protein carriercomprises an amino acid sequence as set forth in SEQ ID NO: 6.

In a preferred embodiment of the invention said composition includes anadjuvant.

In a preferred embodiment of the invention said adjuvant is selectedfrom the group consisting of: cytokines selected from the groupconsisting of e.g. GMCSF, interferon gamma, interferon alpha, interferonbeta, interleukin 12, interleukin 23, interleukin 17, interleukin 2,interleukin 1, TGF, TNFα, and TNFβ.

In a further alternative embodiment of the invention said adjuvant is aTLR agonist such as CpG oligonucleotides, flagellin, monophosphoryllipid A, poly I:C and derivatives thereof.

In a preferred embodiment of the invention said adjuvant is a bacterialcell wall derivative such as muramyl dipeptide (MDP) and/or trehalosedycorynemycolate (TDM).

An adjuvant is a substance or procedure which augments specific immuneresponses to antigens by modulating the activity of immune cells.Examples of adjuvants include, by example only, agonistic antibodies toco-stimulatory molecules, Freunds adjuvant, muramyl dipeptides,liposomes. An adjuvant is therefore an immunomodulator. A carrier is animmunogenic molecule which, when bound to a second molecule augmentsimmune responses to the latter. The term carrier is construed in thefollowing manner. A carrier is an immunogenic molecule which, when boundto a second molecule augments immune responses to the latter. Someantigens are not intrinsically immunogenic yet may be capable ofgenerating antibody responses when associated with a foreign proteinmolecule such as keyhole-limpet haemocyanin or tetanus toxoid. Suchantigens contain B-cell epitopes but no T cell epitopes. The proteinmoiety of such a conjugate (the “carrier” protein) provides T-cellepitopes which stimulate helper T-cells that in turn stimulateantigen-specific B-cells to differentiate into plasma cells and produceantibody against the antigen. Helper T-cells can also stimulate otherimmune cells such as cytotoxic T-cells, and a carrier can fulfil ananalogous role in generating cell-mediated immunity as well asantibodies.

In a preferred embodiment of the invention said composition includes atleast one additional anti-bacterial agent.

In a preferred embodiment of the invention said additionalanti-bacterial agent is a different antigenic molecule.

In a preferred embodiment of the invention said composition is amultivalent antigenic composition.

In an alternative preferred embodiment of the invention said additionalanti-bacterial agent is an antibiotic.

According to a further aspect of the invention there is provided avaccine composition according to the invention for use in the preventionor treatment of a Francisella infection.

Preferably said infection is caused by Francisella tularensis.

According to a further aspect of the invention there is provided amethod to treat a Francisella infection comprising administering aneffective amount of a vaccine or immunogenic composition according tothe invention.

Preferably said infection is caused by Francisella tularensis.

According to an aspect of the invention there is provided an antigenicpolypeptide comprising: a carrier polypeptide comprising one or moreT-cell dependent epitopes and one or more amino acid sequences havingthe amino acid motif D/E-X-N-X-S/T wherein X is any amino acid exceptproline and crosslinked to said carrier polypeptide is an antigenicpolysaccharide wherein the polysaccharide is isolated from Francisellaand is an O-antigen.

In a preferred embodiment of the invention said O-antigen comprises4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-QuiNAc-(1-2)-β-D-Qui4NFm-(1-),where GalNAcAN is 2-acetamido-2-deoxy-O-D-galact-uronamide, 4NFm is4,6-dideoxy-4-formamido-D-glucose and the reducing end group QuiNAc is2-acetamido-2,6-dideoxy-O-D-glucose.

According to a further aspect of the invention there is provided amodified bacterial cell wherein said cell is genetically modified toinclude:

-   -   i) a nucleic acid molecule comprising the nucleotide sequence of        the Francisella O-antigen biosynthetic polysaccharide locus [SEQ        ID NO: 7];    -   ii) a nucleic acid molecule comprising a nucleotide sequence of        an oligosaccharyltransferase [SEQ ID NO: 8] or a functional        variant thereof wherein said variant comprises a nucleic acid        molecule the complementary strand of which hybridizes under        stringent hybridization conditions to the sequence set forth in        SEQ ID NO: 8 and wherein said nucleic acid molecule encodes an        oligosaccharyltransferase; and/or    -   iii) a nucleic acid molecule comprising a nucleotide sequence of        an oligosaccharyltransferase [SEQ ID NO: 16] or a functional        variant thereof wherein said variant comprises a nucleic acid        molecule the complementary strand of which hybridizes under        stringent hybridization conditions to the sequence set forth in        SEQ ID NO: 16 and wherein said nucleic acid molecule encodes an        oligosaccharyltransferase; and    -   iv) a nucleic acid molecule comprising a nucleotide sequence of        carrier polypeptide wherein the a carrier polypeptide comprising        one or more T-cell dependent epitopes and one or more amino acid        sequences having the amino acid motif D/E-X-N-X-S/T wherein X is        any amino acid except proline, wherein said bacterial cell is        adapted for expression of each nucleic acid molecule and        synthesizes an antigenic polypeptide according to the invention.

Hybridization of a nucleic acid molecule occurs when two complementarynucleic acid molecules undergo an amount of hydrogen bonding to eachother. The stringency of hybridization can vary according to theenvironmental conditions surrounding the nucleic acids, the nature ofthe hybridization method, and the composition and length of the nucleicacid molecules used. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed inSambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001); and Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes Part I, Chapter 2(Elsevier, N.Y., 1993). The Tm is the temperature at which 50% of agiven strand of a nucleic acid molecule is hybridized to itscomplementary strand.

The following is an exemplary set of hybridization conditions and is notlimiting.

Very High Stringency (Allows Sequences that Share at Least 90% Identityto Hybridize)

-   -   i) Hybridization: 5×SSC at 65° C. for 16 hours    -   ii) Wash twice: 2×SSC at room temperature (RT) for 15 minutes        each    -   iii) Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Allows Sequences that Share at Least 80% Identity toHybridize)

-   -   i) Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours    -   ii) Wash twice: 2×SSC at RT for 5-20 minutes each    -   iii) Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Allows Sequences that Share at Least 50% Identity toHybridize)

-   -   i) Hybridization: 6×SSC at RT to 55° C. for 16-20 hours    -   ii) Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30        minutes each.

A variant oligosaccharyltransferase polypeptide as herein disclosed maydiffer in amino acid sequence by one or more substitutions, additions,deletions, truncations that may be present in any combination. Amongpreferred variants are those that vary from a reference polypeptide [SEQID NO: 9, SEQ ID NO: 17] by conservative amino acid substitutions. Suchsubstitutions are those that substitute a given amino acid by anotheramino acid of like characteristics. The following non-limiting list ofamino acids are considered conservative replacements (similar): a)alanine, serine, and threonine; b) glutamic acid and aspartic acid; c)asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine,methionine and valine and f) phenylalanine, tyrosine and tryptophan.Most highly preferred are variants that retain or enhance the samebiological function and activity as the reference polypeptide from whichit varies.

In one embodiment, the variant polypeptides have at least 40% or 45%identity, more preferably at least 50% identity, still more preferablyat least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identity, or at least99% identity with the full length amino acid sequence illustratedherein.

In a preferred embodiment of the invention at least theoligosaccharyltransferase of ii) or iii) above is integrated into thebacterial genome to provide a stably transfected and expressingoligosaccharyltransferase.

In a further preferred embodiment of the invention one or more nucleicacid molecules encoding carrier polypeptides are also integrated intothe bacterial genome.

According to a further aspect of the invention there is provided abacterial cell culture comprising a genetically modified bacterial cellaccording to the invention.

According to a further aspect of the invention there is provided aprocess for the production of one or more glycoconjugates comprising:

-   -   i) providing a bacterial cell culture according to the        invention;    -   ii) providing cell culture conditions; and    -   iii) isolating one or more glyconjugates from the bacterial cell        or cell culture medium.

According to a further aspect of the invention there is provided a cellculture vessel comprising a bacterial cell culture according to theinvention.

In a preferred embodiment of the invention said cell culture vessel is afermentor.

Bacterial cultures used in the process according to the invention aregrown or cultured in the manner with which the skilled worker isfamiliar, depending on the host organism. As a rule, bacteria are grownin a liquid medium comprising a carbon source, usually in the form ofsugars, a nitrogen source, usually in the form of organic nitrogensources such as yeast extract or salts such as ammonium sulfate, traceelements such as salts of iron, manganese and magnesium and, ifappropriate, vitamins, at temperatures of between 0° C. and 100° C.,preferably between 10° C. and 60° C., while gassing in oxygen.

The pH of the liquid medium can either be kept constant, that is to sayregulated during the culturing period, or not. The cultures can be grownbatchwise, semi-batchwise or continuously. Nutrients can be provided atthe beginning of the fermentation or fed in semi-continuously orcontinuously. The products produced can be isolated from the bacteria asdescribed above by processes known to the skilled worker, for example byextraction, distillation, crystallization, if appropriate precipitationwith salt, and/or chromatography. In this process, the pH value isadvantageously kept between pH 4 and 12, preferably between pH 6 and 9,especially preferably between pH 7 and 8.

An overview of known cultivation methods can be found in the textbookBioprocess technology 1. Introduction to Bioprocess technology (GustavFischer Verlag, Stuttgart, 1991) or in the textbook by Storhas(Bioreaktoren and periphere Einrichtungen [Bioreactors and peripheralequipment] (Vieweg Verlag, Brunswick/Wiesbaden, 1994)).

The culture medium to be used must suitably meet the requirements of thebacterial strains in question. Descriptions of culture media for variousbacteria can be found in the textbook “Manual of Methods for GeneralBacteriology” of the American Society for Bacteriology (Washington D.C.,USA, 1981).

As described above, these media which can be employed in accordance withthe invention usually comprise one or more carbon sources, nitrogensources, inorganic salts, vitamins and/or trace elements.

Preferred carbon sources are sugars, such as mono-, di- orpolysaccharides. Examples of carbon sources are glucose, fructose,mannose, galactose, ribose, sorbose, ribulose, lactose, maltose,sucrose, raffinose, starch or cellulose. Sugars can also be added to themedia via complex compounds such as molasses or other by-products fromsugar refining. The addition of mixtures of a variety of carbon sourcesmay also be advantageous. Other possible carbon sources are oils andfats such as, for example, soya oil, sunflower oil, peanut oil and/orcoconut fat, fatty acids such as, for example, palmitic acid, stearicacid and/or linoleic acid, alcohols and/or polyalcohols such as, forexample, glycerol, methanol and/or ethanol, and/or organic acids suchas, for example, acetic acid and/or lactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds ormaterials comprising these compounds. Examples of nitrogen sourcescomprise ammonia in liquid or gaseous form or ammonium salts such asammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate or ammonium nitrate, nitrates, urea, amino acids or complexnitrogen sources such as cornsteep liquor, soya meal, soya protein,yeast extract, meat extract and others. The nitrogen sources can be usedindividually or as a mixture.

Inorganic salt compounds which may be present in the media comprise thechloride, phosphorus and sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds such as, for example, sulfates,sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or elseorganic sulfur compounds such as mercaptans and thiols may be used assources of sulfur for the production of sulfur-containing finechemicals, in particular of methionine.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used assources of phosphorus.

Chelating agents may be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents comprisedihydroxyphenols such as catechol or protocatechuate and organic acidssuch as citric acid.

The fermentation media used according to the invention for culturingbacteria usually also comprise other growth factors such as vitamins orgrowth promoters, which include, for example, biotin, riboflavin,thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine.Growth factors and salts are frequently derived from complex mediacomponents such as yeast extract, molasses, cornsteep liquor and thelike. It is moreover possible to add suitable precursors to the culturemedium. The exact composition of the media compounds heavily depends onthe particular experiment and is decided upon individually for eachspecific case. Information on the optimization of media can be found inthe textbook “Applied Microbiol. Physiology, A Practical Approach”(Editors P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN0 19 963577 3). Growth media can also be obtained from commercialsuppliers, for example Standard 1 (Merck) or BHI (brain heart infusion,DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 barand 121° C.) or by filter sterilization. The components may besterilized either together or, if required, separately. All mediacomponents may be present at the start of the cultivation or addedcontinuously or batchwise, as desired.

The culture temperature is normally between 15° C. and 45° C.,preferably at from 25° C. to 40° C., and may be kept constant or may bealtered during the experiment. The pH of the medium should be in therange from 5 to 8.5, preferably around 7.0. The pH for cultivation canbe controlled during cultivation by adding basic compounds such assodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia oracidic compounds such as phosphoric acid or sulfuric acid. Foaming canbe controlled by employing antifoams such as, for example, fatty acidpolyglycol esters. To maintain the stability of plasmids it is possibleto add to the medium suitable substances having a selective effect, forexample antibiotics. Aerobic conditions are maintained by introducingoxygen or oxygen-containing gas mixtures such as, for example, ambientair into the culture. The temperature of the culture is normally 20° C.to 45° C. and preferably 25° C. to 40° C. The culture is continued untilformation of the desired product is at a maximum. This aim is normallyachieved within 10 to 160 hours.

The fermentation broth can then be processed further. The biomass may,according to requirement, be removed completely or partially from thefermentation broth by separation methods such as, for example,centrifugation, filtration, decanting or a combination of these methodsor be left completely in said broth. It is advantageous to process thebiomass after its separation.

However, the fermentation broth can also be thickened or concentratedwithout separating the cells, using known methods such as, for example,with the aid of a rotary evaporator, thin-film evaporator, falling-filmevaporator, by reverse osmosis or by nanofiltration. Finally, thisconcentrated fermentation broth can be processed to obtain the fattyacids present therein.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, means “including but not limited to”, andis not intended to (and does not) exclude other moieties, additives,components, integers or steps. “Consisting essentially” means having theessential integers but including integers which do not materially affectthe function of the essential integers.

Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith.

BRIEF DESCRIPTION OF THE DRAWINGS

An embodiment of the invention will now be described by example only andwith reference to the following figures:

FIG. 1: Principles of Protein Glycan Coupling Technology in E. coli. AnE. coli cell is transformed with three plasmids to generate the clonedglycoconjugate protein (GP). The plasmids encode theoligosaccharyltransferase PgIB, the biosynthetic polysaccharide locusand the carrier protein. The polysaccharide is synthesised on anundecaprenol pyrophosphate lipid anchor (●) within the cytoplasm, thisis transferred to the periplasmic compartment where PgIB recognises thelipid link reducing end sugar and transfers the polysaccharide en bloconto an acceptor sequon (D/E-X-N-X-S/T) on the carrier protein toproduce the glycoconjugate protein (GP). IM, inner membrane; OM, outermembrane;

FIG. 2: F. tularensis SchuS4 O-antigen is expressed in E. coli DH5αcells. E. coli cells carrying the F. tularensis vector pGAB1 (containingO-antigen coding region) and empty pGEM-T Easy vector respectively,stained with DAPI to visualise nucleic acid in blue (panels a and b) andprobed with mAb FB11 and Alexa Fluor® 488 conjugated secondary antibody(panels c and d). Images are shown at 100× magnification;

FIG. 3: F. tularensis O-antigen is conjugated to ExoA by CjPgIB in E.coli CLM24 cells. Two colour immunoblots performed on HIS tag purifiedExoA using mouse mAbFB11 (green) and rabbit anti 6×HIS tag antibody(red). Lane 1, E. coli CLM24 carrying pGAB2, pGVXN150, pGVXN115(non-functional CjPgIB control); lane 2, pGAB2, pGVXN150, pGVXN114(functional CjPgIB); lane 3, pGVXN150 only; Panel 2b, close up view ofHIS-tagged purified ExoA attached to various chain lengths of F.tularensis O-antigen. M=marker IRDye® 680/800 protein marker. pGVXN150contains ExoA, pGAB2 contains F. tularensis O-antigen, pGVXN114 andpGVXN115carry a functional and non-functional CjPgIB respectively;

FIG. 4: Vaccination with test glycoconjugate increases host survivalcompared to LPS and controls. Balb/C mice were vaccinated with threedoses of 10 ug glycoconjugate+SAS, LPS+SAS or 10 μg LPS (n=10 pergroup). Mice were challenged 5 weeks following final vaccination with100 CFU of F. tularensis strain HN63 via the i.p. route. Both LPS,LPS+SAS and test glycoconjugate provided improved protection whencompared to the relevant unvaccinated controls (P<0.001) and the SASalone provided no survival benefit (P>0.05) as analysed by stratifiedlog rank test. The test glycoconjugate provided significantly betterprotection than the LPS alone or LPS+SAS vaccine (P<0.001 and P=0.025respectively).

FIG. 5: Mice vaccinated with test glycoconjugate shows a reducedbacterial load in spleens compared to LPS and controls. Unvaccinated,SAS vaccinated, 10 μg LPS, or 10 μg test glycoconjugate+SAS vaccinatedmice were challenged with 100 CFU of F. tularensis strain HN63 via thei.p. route. Spleens were removed 3 days post infection from each group(n=5) and assessed for bacterial CFUs. Logarithm data were analysedusing a general linear model and Bonferroni's post tests. There was nodifference in bacterial load between SAS vaccinated and unvaccinatedmice (P>0.05) but the 10 μg LPS or 10 μg test glycoconjugatevaccinations had significantly decreased bacterial load when compared torelevant controls (P<0.001). Mice vaccinated with the testglycoconjugate+SAS had significantly reduced bacterial numbers in thespleen compared to LPS (P<0.05).

FIG. 6: Reduced inflammatory responses seen in LPS and glycoconjugatevaccinated mice compared to controls. Unvaccinated, SAS vaccinated, 10μg LPS or 10 μg test glycoconjugate vaccinated mice were challenged with100 CFU of F. tularensis strain HN63 via the i.p. route. Spleens wereremoved 3 days post infection from each group (n=5) and assessed forcytokine response. Levels of IL-6, MCP-1 and IFN-γ, were measures byCBA; all cytokine data pg/spleen. Individual points represent individualsamples with line indicating the mean. Logarithm data was analysed usinga general linear model and Bonferroni's post tests. Cytokine production(IL-6, MCP-1 and IFN-γ) was comparable between controls (untreated andSAS) and the two vaccine treated groups (LPS and glycoconjugate).Cytokine concentration was reduced in vaccinated mice compared torelevant controls (P<0.05) and the experiments 1 and 2 did not differfrom each other (P>0.05);

FIG. 7: Increased IgG response in glycoconjugate vaccinated mice animals7 days prior to challenge. Increased LPS specific IgG was observed inthe glycoconjugate+SAS vaccinated group when compared to animalsvaccinated with LPS only (P<0.001);

FIG. 8: Pilot study of vaccine candidates and relevant controls. Balb/Cmice were vaccinated with three doses, 2 weeks apart with candidatevaccine or relevant controls (n=10 per group). Mice were challenged 5weeks following final vaccination with 100 CFU of F. tularensis strainHN63 via the i.p. route. 0.5 of product per time point were assessed.Mice vaccinated with 0.5 μg test glycoconjugate with SAS (P<0.05) andthe 0.5 μg LPS vaccines (P<0.001) survived longer than controls asdetermined by log rank test. Glycoconjugate, F. tularensis O-antigenExoA glycoconjugate; sham glycoconjugate, C. jejuni 81116heptasaccharide ExoA glycoconjugate; and

FIG. 9: F. tularensis LPS specific IgM levels observed in vaccinatedmice 1 day prior to challenge. There were no differences between LPSspecific IgM levels in the glycoconjugate and SAS vaccinated group whencompared to animals vaccinated with LPS only group (P>0.05). We observedno evidence of the LPS-specific IgM titres differing between experiments(P>0.05).

FIG. 10 (SEQ ID 1) Amino acid sequence of carrier protein ExoA(Pseudomonas aeruginosa)

FIG. 11 (SEQ ID NO: 2) Amino acid sequence of carrier protein TUL4(Francisella tularensis)

FIG. 12 (SEQ ID NO: 3) Amino acid sequence of carrier protein FTT1713c(Francisella tularensis)

FIG. 13 (SEQ ID NO: 4) Amino acid sequence of carrier protein FTT1695(Francisella tularensis)

FIG. 14 (SEQ ID NO: 5) Amino acid sequence of carrier protein FTT1269c(Francisella tularensis)

FIG. 15 (SEQ ID NO: 6) Amino acid sequence of carrier protein FTT1696(Francisella tularensis)

FIGS. 16A-16F (SEQ ID NO: 7) Nucleotide sequence encoding theFrancisella O-antigen biosynthetic polysaccharide

FIG. 17: SEQ ID NO: 8 nucleotide sequence encoding theoligosaccharyltransferase PgIB (C. jejuni)

FIG. 18: SEQ ID NO: 9 Pgl B amino acid sequence (C. jejuni)

FIG. 19: SEQ ID NO: 16 nucleotide sequence encoding theoligosaccharyltransferase PgIB (Campylobacter sputorum)

FIG. 20 SEQ ID NO: 17 Amino acid sequence (full length) of PgIB(Campylobacter sputorum)

FIG. 21 HIS tag purified DNAK protein from cultures of Escherichia coliCLM24. Comparison of 2 plasmid system (chromosomally encoded PgIB) andstandard 3 plasmid system. Lanes 1/3/5, three biological replicates ofDNAK purified from the two plasmid system; Lanes 2/4/6, three biologicalreplicates of DNAK purified from the 3 plasmid system. Three plasmidsystem consists of pGAB2 coding for Francisella tularensis O antigen,pEC415DNAK coding for F. tularensis DNAK and pGVXN114 coding for PgIB.

FIG. 22: The F. tularensis O-antigen is conjugated to ExoA. Treatment ofthe glycoconjugate with proteinase K to degrade ExoA results in loss ofthe O-antigen ladder at the corresponding size. Western blot wasperformed with monoclonal antibody FB11. A, Markers; B, proteinase Kdigested ExoA F. tularensis O-antigen glycoconjugate; C, glycoconjugateheated to 50° C. o/n without proteinase K. S1.2; Silver stained ExoA F.tularensis O-antigen glycoconjugate.

FIG. 23: Comparison of LPS-specific IgM levels from the glycoconjugateand LPS vaccine groups. Panel a, combined anti glycan and anti HISsignal; panel b, anti HIS signal only; panel c, anti-glycan signal only.Lane 1, ₂₆₀DNNNS₂₆₄ altered to ₂₆₀DNQNS₂₆₄; Lane 2, ₄₀₂DQNRT₄₀₆ alteredto ₄₀₂DQQRT₄₀₆; Lane 3, ₂₆₀DNNNS₂₆₄ altered to ₂₆₀DNQNS₂₆₄ and₄₀₂DQNRT₄₀₆ altered to ₄₀₂DQQRT₄₀₆; Lane 4, exotoxin A encoded frompGVXN150.

DETAILED DESCRIPTION

TABLE 1 Strains and plasmids used Strain/plasmid Description Source E.coli DH5α F-φ80lacZΔM15 Invitrogen Δ(lacZYA-argF) U169 deoRrecA1 endA1hsdR17 (rk−, mk+), gal- phoAsupE44λ - thi-1 gyrA96 relA1 E. coli XL-1endA1 gyrA96(nalr)thi-1 Stratagene relA1 lac gln V44 F′[::Tn10 proAB+laclq Δ (lacZ)M15] hsdR17(r_(k) ⁻m_(k) ⁺) E. coli CLM24 rph-lIN(rrnD-rrnE) 1, 5 ΔwaaL F. tularensis subs. Type A strain DSTL, Portontularensis strain SchuS4 Down laboratories F. tularensis subs. Type Bstrain, isolated in Green, M., et al., holarctica strain HN63 Norwayfrom an infected Efficacy of the live attenuated Hare Francisellatularensis vaccine (LVS) in a murine model of disease. Vaccine, 2005.23(20): p. 2680-6 pGEM-T Easy TA cloning vector, amp^(r) Promega pLAFR1Low copy expression Vanbleu E, Marchal K, vector, tet^(r) VanderleydenJ. Genetic and physical map of the pLAFR1 vector. DNA Seq. 2004 June;15(3): 225-7. pGAB1 F. tularensis O antigen This study coding regioninserted into MCS of pGEM-T easy pGAB2 F. tularensis subs. This studytularensis strain SchuS4 O antigen coding region inserted into Ecorlsite of pLAFR. pGVXN114 Expression plasmid for GlycoVaxyn CjPglBregulated from the Lac promoter in pEXT21. IPTG inducible, HA tag,Spec^(r). pGVXN115 Expression plasmid for GlycoVaxyn C. jejuninonfunctionalPglB due to a mutation at ₄₅₇WWDYGY₄₆₂ to ₄₅₇WAAYGY₄₆₂,regulated from the Lac promoter in pEXT21. IPTG inducible, HA tag,Spec^(r). pGVXN150₂₆₀DNQNS₂₆₄ Expression plasmid for This studyPseudomonas aeruginosa PA103(DSM111/) Exotoxin A with the signal peptideof the E.coliDsbA protein, two inserted bacterial N-glycosylation sites,AA at position 262 altered from N to Q and a hexahis tag at theC-terminus. Induction under control of an arabinose inducible promoter,Amp^(r) pGVXN150₄₀₂DQQRT₄₀₆ Expression plasmid for This studyPseudomonas aeruginosa PA103(DSM111/) Exotoxin A with the signal peptideof the E. coliDsbA protein, two inserted bacterial N-glycosylationsites, AA at position 404 altered from N to Q and a hexahis tag at theC-terminus. Induction under control of an arabinose inducible promoter,Amp^(r) pGVXN150₂₆₀DNQNS₂₆₄/ Expression plasmid for This study₄₀₂DQQRT₄₀₆ Pseudomonas aeruginosa PA103(DSM111/) Exotoxin A with thesignal peptide of the E. coliDsbA protein, two inserted bacterialN-glycosylation sites, AA at position 262 and 404 altered from N to Qand a hexahis tag at the C-terminus. Induction under control of anarabinose inducible promoter, Amp^(r) pACYCpgl pACYC184 carrying the 5CjPglB locus, Cm^(r) E. coli CedAPglB E. coli strain CLM24 with a Thisstudy chromosomally inserted IPTG inducible copy of PglBMaterials and MethodsBacterial Strains and Plasmids

Escherichia coli strains were grown in LB at 37° C., 180 r.p.m.Antibiotics were used at the following concentrations; tetracycline 20μg/ml, ampicillin 100 μg/ml, spectinomycin 80 μg/ml, chloramphenicol 30μg/ml. The host strain for initial cloning experiments was E. coli XL-1,subsequent strains used for glycoconjugate production were E. coli DH5αand CLM24 ( Table 1). For efficacy studies, mice were challenged with F.tularensis subsp. holarctica strain HN63. The bacterium was cultured onblood cysteine glucose agar plates (supplemented with 10 ml of 10%(wt/vol) histidine per litre) at 37° C. for 18 hours.

Cloning, Sequencing and Expression of the F. tularensis O Antigen CodingRegion

DNA was prepared from the F. tularensis subsp. tularensis strain SchuS4by phenol extraction as described by Karlsson et al. Microb CompGenomics. 5(1):25-39, (2000). The O-antigen coding region was amplifiedusing the primers FTfragment2rev (5′-GGATCATTAATAGCTAAATGTAGTGCTG-3′;SEQ ID 10) and Oantlftfwd (5′-TTTTGAATTCTACAGGCTGTCAATGGAGAATG-3′; SEQID 11) using the following cycling conditions: 94° C., 15 sec, 55° C.,15 sec, 68° C., 20 min; 35 cycles using Accuprime TaqHifi (InvitrogenU.K.). This was cloned into the TA cloning vector pGEM-T Easy togenerate the vector pGAB1. The plasmid pGAB1 was digested with EcoRI andthe insert was subcloned into the vector pLAFR to generate the constructpGAB2.

Immunofluorescence Imaging of E. coli Cells Carrying F. tularensis OAntigen Coding Region

Immunofluorescence was carried out as previously described [17] with themodification that the IgG2a mouse monoclonal antibody FB11 was used todetect F. tularensis O antigen (1 μl/ml in 5% (v/v) FCS/PBS).

Production and Purification of Glycoconjugate Vaccine

E. coli CLM24 carrying the vectors pGAB2, pGVXN114 and pGVXN150 wasgrown for 16 h in 200 mL LB broth at 37° C., 180 r.p.m. This was used toinoculate 1.8 L of LB broth and further incubated at 110 r.p.m. 37° C.to an OD.600 nm reading of 0.4 to 0.6. L-arabinose was then added to afinal concentration of 0.2% and IPTG to a final concentration of 1 mM toinduce expression of exoA and CjpglB respectively; following 5 hours ofincubation, 0.2% L-arabinose was added again and the culture left toincubate o/n.

Cells were harvested by centrifugation at 6,000 r.p.m. for 30 m, andpelleted cells were incubated at room temperature for 30 m in a lysissolution composed of 10× BugBuster protein extraction reagent (Novagen)diluted to 1× in 50 mM NaH2PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0supplemented with 0.1% Tween, 1 mg/ml lysozyme and 1 μl/ml Benzonasenuclease (Novagen). Cell debris was removed by centrifugation at 10,000r.p.m. for 30 m, the supernatant was collected and 1 ml Ni-NTA agarose(QIAgen) was added to the supernatant. The slurry-lysate was incubatedfor 1 h at 4° C. with shaking then loaded into 10 ml polypropylenecolumns (Thermo scientific). His tagged ExoA was purified according tomanufacturer's instructions (QIA expressionist, QIAGEN) with theaddition of 20% glycerol and 5% glucose to the elution buffer. Proteinyields were estimated using a bicinchonic acid assay kit according tomanufacturer's instructions (Pierce® Biotechnology BCA protein AssayKit, U.S.A.).

For large-scale protein purification, material was isolated using GEHealthcare His Trap columns and an AKTA purifier with an imidazolegradient of 30-500 mM. The collected fraction containing ExoAglycosylated with F. tularensis O-antigen was further purified using aresource Q anionic exchange column (GE Healthcare) with a NaCl gradientfrom 0 to 500 mM in 20 mM TrisHCl pH 8.0. This generated a typical yieldof 2-3 mg/ml of glycoconjugate per 2 L of E. coli culture.

The same techniques were used for the generation of the ‘sham’ C. jejuniheptasaccharide ExoA glycoconjugate encoded by pACYCpgl [18].

Using the E. coli chromosomally inserted strain CLM 24 CedAPglB:Escherichia coli strain CLM24 with a chromosomally inserted copy of pgIBwere grown in Luria-Bertani (LB) broth at 37° C., with shaking.Antibiotics were used at the following concentrations: tetracycline 20μg ml⁻¹ and ampicillin 100 μg ml⁻¹. Tetracycline was used to maintainthe plasmid pGAB2 coding for Francisella tularensis O antigen andampicillin was used to maintain the plasmid coding for the acceptorcarrier protein.

Escherichia coli cells were grown for 16 h in 200 ml LB broth at 37° C.,with shaking. This was used to inoculate 1.8 l of LB broth and furtherincubated with shaking at 37° C. until an OD600 reading of 0.4-0.6 wasreached. At this point L-arabinose was added to a final concentration of0.2 per cent and IPTG to a final concentration of 1 mM to induceexpression of the acceptor protein and pgIB, respectively; after another5 h of incubation, 0.2% L-arabinose was added again and the culture leftto incubate overnight.

Cells were harvested by centrifugation at 5300 g for 30 min, andpelleted cells were incubated at room temperature for 30 min in a lysissolution composed of 10× BugBuster protein extraction reagent (Novagen)diluted to 1× in 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0supplemented with 0.1 per cent Tween, 1 mg ml⁻¹ lysozyme and 1 μl ml⁻¹Benzonase nuclease (Novagen). Cell debris was removed by centrifugationat 7840 g for 30 min, the supernatant was collected and 1 ml Ni-NTAagarose (QIAGEN) was added to the supernatant. The slurry-lysate wasincubated for 1 h at 4° C. with shaking then loaded into 10 mlpolypropylene columns (Thermo Scientific). His-tagged ExoA was purifiedby the addition of an elution buffer according to manufacturer'sinstructions (QIA expressionist, QIAGEN) containing 250 mM imidazolewith the addition of 20 per cent glycerol and 5 per cent glucose.

Alternatively cells were grown in LB agar plates containingtetracycline, ampicillin, IPTG to a final concentration of 50 μM andL-arabinose to a final concentration of 0.2% for 16 h at 37 ° C. Cellswere subsequently harvested by scraping and protein purified asindicated above.

Immunoblot Analysis

To verify transfer and presence of the F. tularensis O antigen, sampleswere analysed by western blotting. E. coli cells were grown o/n in 10 mlLB broth and diluted to an O.D.600 nm of 1.0. Cells were centrifuged at13,000 r.p.m. for 10 min, supernatant was removed and cells wereresuspended in 100 μl Laemmli buffer and lysed by boiling for 10 minbefore analysis by western blotting or silver staining. Mouse anti F.tularensis O-antigen monoclonal antibody FB011 (AbCam U.K.) was used ata dilution of 1:1,000, rabbit anti HIS monoclonal antibody was used todetect ExoA at a dilution of 1:10,000 (AbCam U.K.). Secondary antibodiesused were goat anti mouse IRDye680 and IRDye800 conjugates used at1:5000 dilutions. Signal detection was undertaken using the Odyssey®LI-COR detection system (LI_COR Biosciences GmbH).

Cytokine Response Analysis

Spleen supernatants were assessed using mouse inflammatory cytometricbead array kit (CBA-BD biosciences) for IL-10, IL-12p70, IFN-γ, IL-6,TNF-α, and MCP-1. Samples were incubated with the combined capture beadcocktail, and incubated for 1 h at room temperature. Followingincubation, PE detection antibodies were added and incubated for afurther 1 h. Samples were then washed and resuspended in FACS buffer.Cytokine concentrations were measured via quantification of PEfluorescence of samples in reference to a standard curve.

BALB/c Mouse Challenge Studies

Female Balb/C mice were obtained from Charles River Laboratories (Kent,U.K.) at 6-8 weeks of age. The pilot study was done in groups of 10 miceimmunised with either 0.5 μg F. tularensis LPS, 0.5 μg F. tularensisglycoconjugate, 0.5 μg F. tularensis glycoconjugate +SAS, 0.5 μg ‘sham’glycoconjugate +SAS, 0.5 μg ‘sham’ glycoconjugate or SAS only. One groupof mice were left untreated as challenge efficacy controls.Immunisations occurred on days 0, 14 and 28 via intra-peritoneal (IP)route. Mice were challenged 35 days post-immunisation with 100 CFU of F.tularensis strain HN63 by the IP route, delivered in 0.1 ml. Subsequentexperiments used the same schedule with 15 mice per group and doses of10 μg of material per immunisation. Four weeks following finalvaccination 5 mice from each group were tail bled to obtain sera forantibody analysis and culled at day 3 post-infection with spleensharvested to analyse bacterial load and cytokine response. For theenumeration of bacteria, spleen samples were homogenized in 2 ml of PBSthrough 40 μm cell sieves (BD Biosciences) and 100 μl aliquots wereplated onto BCGA plates. F. tularensis LPS-specific IgM and total IgGlevels were determined by ELISA as previously described [19]. All workwas performed under the regulations of the Home Office ScientificProcedures Act (1986).

Statistical Analysis

Statistical analyses were performed using the program PASW (SPSS release18.0). Survival data was analysed by pair-wise Log Rank test stratifiedby experiment. Cytokine and bacterial load data were analysed usingunivariate general linear models, using Bonferroni's post tests tofurther clarify significant differences.

Production and Purification of Glycoconjugate Vaccine

E. coli CLM24 carrying the vectors pGAB2, pGVXN114 and pGVXN150 wasgrown for 16 h in 200 mL LB broth at 37° C., 180 r.p.m. This was used toinoculate 1.8 L of LB broth and further incubated at 110 r.p.m. 37° C.until an O.D600 reading of 0.4 to 0.6 was reached. At this pointL-arabinose was added to a final concentration of 0.2% and IPTG to afinal concentration of 1 mM to induce expression of exoA and CjpglBrespectively; after another 5 hours of incubation, 0.2% L-arabinose wasadded again and the culture left to incubate overnight.

Cells were harvested by centrifugation at 6,000 r.p.m. for 30 m, andpelleted cells were incubated at room temperature for 30 m in a lysissolution composed of 10× BugBuster protein extraction reagent (Novagen)diluted to 1× in 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0supplemented with 0.1% Tween, 1 mg/ml lysozyme and 1 μl/ml Benzonasenuclease (Novagen). Cell debris was removed by centrifugation at 10,000r.p.m. for 30 m, the supernatant was collected and 1 ml Ni-NTA agarose(QIAgen) was added to the supernatant. The slurry-lysate was incubatedfor 1 h at 4° C. with shaking then loaded into 10 ml polypropylenecolumns (Thermo scientific). His tagged ExoA was purified by theaddition of an elution buffer according to manufacturer's instructions(QIA expressionist, QIAGEN) containing 250 mM imidazole with theaddition of 20% glycerol and 5% glucose. Protein yields were estimatedusing a bicinchonic acid assay kit according to manufacturer'sinstructions (Pierce® Biotechnology BCA protein Assay Kit, U.S.A.).

For large-scale protein purification, material was isolated using GEHealthcare HIS trap columns and an AKTA purifier with an imidazolegradient of 30 mM to 500 mM. The collected fraction containing ExoAglycosylated with F. tularensis O-antigen was further purified using aresource Q anionic exchange column (GE Healthcare) with a NaCl gradientfrom 0 to 500 mM in 20 mM TrisHCl pH 8.0. This generated a typical yieldof 2-3 mg/ml of glycoconjugate per 2 L of E. coli culture.

The same techniques were used for the generation of the ‘sham’ C. jejuniheptasaccharide ExoA glycoconjugate. The plasmid coding for thisheptasaccharide was pACYCpgl carrying the entire Cjpgl cluster from C.jejuni 81116 [1].

Protein Expression

Escherichia coli strain CLM24 with a chromosomally inserted copy of pglBwere grown in Luria-Bertani (LB) broth at 37° C., with shaking.Antibiotics were used at the following concentrations: tetracycline 20μg ml-1 and ampicillin 100 μg ml-1. Tetracycline was used to maintainthe plasmid pGAB2 coding for Francisella tularensis O antigen andampicillin was used to maintain the plasmid coding for the acceptorcarrier protein.

Escherichia coli cells were grown for 16 h in 200 ml LB broth at 37° C.,with shaking. This was used to inoculate 1.8 l of LB broth and furtherincubated with shaking at 37° C. until an OD600 reading of 0.4-0.6 wasreached. At this point L-arabinose was added to a final concentration of0.2 per cent and IPTG to a final concentration of 1 mM to induceexpression of the acceptor protein and pglB, respectively; after another5 h of incubation, 0.2 per cent L-arabinose was added again and theculture left to incubate overnight.

Cells were harvested by centrifugation at 5300 g for 30 min, andpelleted cells were incubated at room temperature for 30 min in a lysissolution composed of 10× BugBuster protein extraction reagent (Novagen)diluted to 1× in 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0supplemented with 0.1 per cent Tween, 1 mg ml-1 lysozyme and 1 μl ml-1Benzonase nuclease (Novagen). Cell debris was removed by centrifugationat 7840 g for 30 min, the supernatant was collected and 1 ml Ni-NTAagarose (QIAGEN) was added to the supernatant. The slurry-lysate wasincubated for 1 h at 4° C. with shaking then loaded into 10 mlpolypropylene columns (Thermo Scientific). His-tagged ExoA was purifiedby the addition of an elution buffer according to manufacturer'sinstructions (QIA expressionist, QIAGEN) containing 250 mM imidazolewith the addition of 20 per cent glycerol and 5 per cent glucose.

EXAMPLES Example 1 Expression of the F. tularensis SchuS4 O-antigen inE. coli DH5α Cells

The 20 kb F. tularensis SchuS4 O-antigen coding region was PCR amplifiedand cloned into pGEM-T Easy to generate the plasmid pGAB1. All bacterialstrains and vectors used in this study are summarized in table 1. Toconfirm O-antigen expression and transport to the outer cell surface ofE. coli, pGAB1 was transformed into DH5α cells and probed byimmunofluorescence using mAb FB11, specific to the F. tularensisO-antigen. FIG. 2C demonstrates the expression of the O-antigen on thesurface of E. coli DH5a cells, which is absent in the vector alonecontrol (FIG. 2D).

Example 2 CjPgIB can Transfer F. tularensis O-antigen to the AcceptorProtein Exotoxin A

In order to generate a strong T-cell response and lasting immunity, ahighly immunogenic protein is required as a carrier for the F.tularensis O-antigen. The selected carrier protein was an inactivatedform of the P. aeruginosa Exotoxin A variant L552V, AE553 (ExoA) wasselected [20]. The plasmid pGAB2 containing the F. tularensis O-antigenexpressed in the low copy vector pLAFR1 [21] was transformed into E.coli CLM24 cells along with the plasmids pGVXN114 and pGVXN150 whichcontain CjPgIB and ExoA respectively. As negative glycosylationcontrols, CLM24 cells were transformed with either pGVXN150 alone orwith the combination of pGAB2, pGVXN150 and pGVXN115, the latter codingfor an inactive version of CjPgIB [18]. Following overnight induction ofCjpglB and exoA expression with 1 mM IPTG and 0.2% L-arabinose (w/v)respectively, cells were lysed and HIS tagged ExoA purified using Nickelcolumns. Elution fractions from each sample were separated by SDS PAGEand tested by immunoblotting with mAbFB011 specific for F. tularensisLPS. A band matching the expected size of ExoA and an O-antigen ladderpattern could only be seen when a functional CjPgIB was present (FIG. 3,lanes 2 and 2b). In the absence of a functional CjPgIB there was nocross-reaction with mAbFB11 (FIG. 3, lanes 1 and 3). To demonstrate thatthe O-antigen was bound to the carrier protein, HIS tagged ExoA F.tularensis O-antigen conjugate was purified and digested with ProteinaseK. The disappearance of the O-antigen ladder after Proteinase Ktreatment but not in the untreated control confirmed that the O-antigenwas anchored to ExoA (data not shown).

Example 3 Vaccination with the Glycoconjugate Provides SignificantProtection against F. tularensis subsp. holarctica Infection in Mice

In a pilot study we compared LPS alone against the glycoconjugatevaccine and monitored antibody levels and murine survival. The SigmaAdjuvant System® was selected for use in this study because it is basedon monophosphoryl lipid A (MPL), a low toxicity derivative of LPS thathas been demonstrated to be a safe and effective immunostimulant [22].In order to demonstrate the specificity of the glycoconjugate we usedcontrols including mice with SAS adjuvant alone, unvaccinated mice andmice vaccinated with a ‘sham’ glycoconjugate control (C. jejuniheptasaccharide conjugated to ExoA). Only mice vaccinated with 0.5 μgtest glycoconjugate+SAS (P<0.05) or 0.5 μg LPS (P<0.001) demonstratedincreased survival compared to the appropriate controls as determined bylog rank test (FIG. 22). These candidates were selected for furtherassessment at higher doses and an additional group consisting of LPS+SASwas also added as a further control. Protection was compared betweenmice immunised with either 10 μg glycoconjugate+SAS, 10 μg LPS or 10 μgLPS+SAS. All three vaccines were protective when compared to theunvaccinated mice (P<0.001), while the SAS adjuvant alone did not elicitany protection (P>0.05) (FIG. 4). This experiment also indicated thatLPS+SAS did not elicit the same level of protection as theglycoconjugate+SAS combination (P<0.05) and thereafter LPS+SAS wasdeemed unnecessary for testing. The study was repeated in order toprovide further bacterial organ load and immunological response data andno statistically significant difference was found between replicates.

Example 4 Mice Vaccinated with Test Glycoconjugate and Challenged withF. tularensis subsp. holarctica have Lower Bacterial Loads andPro-Inflammatory Cytokines 3 Days Post Challenge

Three days post challenge 5 mice per group were sacrificed and bacterialloads in the spleens and inflammatory responses were evaluated (FIG. 5).FIG. 5 shows the bacterial loads from vaccine with 10 μg of eachcandidate. Mice that were immunised with the glycoconjugate+SAS or LPSboth had significantly decreased bacterial loads in spleens (P<0.01)when compared to the SAS and unvaccinated controls. Mice vaccinated withglycoconjugate+SAS had significantly less bacteria compared to thosevaccinated with LPS alone (P<0.05). Inflammatory cytokine profilesbetween the different vaccine groups were also analysed (FIG. 6).Reduced levels of inflammatory cytokines were seen in mice vaccinatedwith glycoconjugate+SAS and LPS alone (P<0.05), corresponding withdecreased bacterial loads. There was no significant difference betweencytokine profiles for both experiments (P>0.05).

Example 5 Vaccination with the F. tularensis Glycoconjugate Induces aGreater IgG Immune Response

The levels of LPS-specific IgG were assessed in mice 7 days prior tochallenge for both experiments. Increased LPS-specific IgG was observedin the glycoconjugate+SAS vaccinated group when compared to animalsvaccinated with LPS only (P<0.001). Although experiment 2 had higherlevels of antibody (P<0.01), we observed no difference in patternbetween experiments (P>0.05) (FIG. 7). No significant differences wereobserved between LPS-specific IgM levels from the glycoconjugate and LPSvaccine groups (FIG. 23).

REFERENCES

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The invention claimed is:
 1. A process for manufacturing aglycoconjugate polypeptide, comprising: i) providing a bacterial cell incell culture medium, wherein said bacterial cell is genetically modifiedto include: a) a nucleic acid molecule comprising the nucleotidesequence of the Francisella O-antigen biosynthetic polysaccharide locusset forth in SEQ ID NO: 7; b) a nucleic acid molecule comprising theoligosaccharyltransferase nucleotide sequence set forth in SEQ ID NO: 8or a functional variant thereof, wherein said functional variantcomprises a nucleic acid molecule the complementary strand of whichshares at least 90% identity to the sequence set forth in SEQ ID NO: 8and wherein said nucleic acid molecule encodes anoligosaccharyltransferase; and c) a nucleic acid molecule comprising anucleotide sequence of a carrier polypeptide, wherein the carrierpolypeptide comprises one or more T-cell dependent epitopes and one ormore amino acid sequences having the amino acid motif D/E-X-N-X-S/T,wherein X is any amino acid except proline; wherein the bacterial cellis adapted for expression of each nucleic acid molecule set forth in a),b), and c); ii) growing the bacterial cell in the cell culture medium,wherein the oligosaccharyltransferase of b) glycosylates the carrierpolypeptide of c) to form a glycoconjugate polypeptide comprising aFrancisella O-antigen; and iii) isolating the glycoconjugate polypeptidecomprising a Francisella O-antigen from the bacterial cell or the cellculture medium, thereby manufacturing a glycoconjugate polypeptide. 2.The process according to claim 1, wherein at least theoligosaccharyltransferase is integrated into the bacterial genome ofsaid bacterial cell.
 3. The process according to claim 1, wherein one ormore nucleic acid molecules encoding the carrier polypeptide isintegrated into the bacterial genome of said bacterial cell.
 4. Theprocess according to claim 1, wherein said Francisella O-antigencomprises 4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-QuiNAc-(1-2)-α-D-Qui4NFm-(1-), wherein GalNAcAN is-2-acetamido-2-deoxy-O-D-galact-uronamide, 4NFm is4,6-dideoxy-4-formamido-D-glucose and the reducing end group QuiNAc is2-acetamido-2,6-dideoxy-O-D-glucose.
 5. The process according to claim4, wherein said Francisella O-antigen is a tetrasaccharide.
 6. Aglycoconjugate polypeptide obtained by the process according to claim 1.7. A vaccine or immunogenic composition comprising the glycoconjugatepolypeptide according to claim 6 and an adjuvant and/or carrier.
 8. Amethod for treating a Francisella infection, comprising administering aneffective amount of the vaccine or immunogenic composition according toclaim
 7. 9. The method according to claim 8, wherein saidFrancisellainfection is caused by Francisella tularensis.